Arrays of immobilized probes are currently being developed for use in assays to detect and identify components in biological samples and for screening molecular libraries. The ability to screen for multiple species of molecules in a single assay test is particularly valuable for purposes of drug discovery and clinical genetics. Accordingly, array manufacturing technologies have been developed to permit a large number of different probes to be incorporated into an array at separate and known locations (see, e.g., Fodor et al., Science 251, 767-773 (1991); Southern et al., Nucleic Acids Research 22: 1368-1373 (1994); U.S. Pat. No. 5,510,270; U.S. Pat. No. 5,474,796; U.S. Pat. No. 5,429,807; and U.S. Pat. No. 5,472,672). In these prior art methods, the probe molecules are synthesized in situ on a solid support surface at predetermined locations.
There are disadvantages to forming an array in this manner when the array is comprised of biopolymers such as oligonucleotides. The in situ method currently used for the commercial manufacture of oligonucleotide arrays is not well suited for the efficient production of arrays of long chain polymer probes because of the number of cycles of elongation required and the variable efficiency of each attachment step. For example, in order to synthesize n species of oligonucleotide having m variable positions, n×m elongation cycles are required. Although solutions have been devised to shorten the time of the overall process by segregating groups of sites for the simultaneous addition of a given nucleotide (see, e.g., Frank et al., Nucleic Acids Res. 11, 4365-4377 (1983); U.S. Pat. No. 5,510,270), manufacturing an array in this way is inefficient, particularly when the desired array is intended to include hundreds of different probe sequences and probe lengths in excess of 30 nucleotides. Each attachment step requires a finite time for covalent bond formation, and each is associated with a failure rate of between 2% and 15%. The problems with probe fidelity necessitate a high level of probe redundancy, and it is only after the entire array is formed that defects are discoverable.
Additionally, if reagents are dispensed as microdroplets, precautions must be taken to avoid intermixing of chemical moieties in neighboring droplets either by precisely depositing the droplet at its designated attachment site or by forming a pattern of differential polarities on the attachment surface to constrain the droplets to their designated attachment regions by means of surface tension.
What is needed is a process for forming an array with probes of known high fidelity in which the chemistry of attachment can be independently optimized for each different probe in the array, the density of each type of attached probe can be quantitatively assessed prior to array formation and probes not meeting specifications can be corrected or discarded prior to array assembly. The number of overall steps required to form the array is reduced in this process compared to prior art methods, and the total number of tests required to ensure the quality of manufactured arrays is equal to the number of probes in the array, not the number of arrays produced.